Impaired circulation is an underlying aspect of many clinically-manifested disorders including peripheral arterial disease (PAD), ischemic heart disease and chronic wounds. More than 24 million patients are afflicted with these conditions in the US, with 10 million affected by PAD alone. PAD is often a result of diabetes, which currently affects one in three adults over the age of 40 and whose incidence is expected to increase as a result of increasing obesity in the general population. Chronic wounds affect more than 6.5 million patients each year and cause significant impairment of quality of life. Even with current treatments, about 35,000 limb amputations are performed each year due to life-threatening ischemia.
Wound healing after injury occurs in three major stages. First is the hemostatic and inflammatory stage, which minimizes blood loss and recruits specific cells to the site of injury. Platelets engage the injured tissue, initiate clot formation and release growth factors during this first stage. In the second stage, recruited phagocytic cells such as macrophages and monocytes digest the injured tissue and angiogenic growth factors released by activated platelets, macrophages, and other cells bind to receptors on the surface of endothelial cells in pre-existing blood vessels. The endothelial cells then proliferate, migrate into a wound bed, and differentiate into arterial and venous vascular tissue. Finally, in a third remodeling stage, new blood vessels mature by recruiting smooth muscle cells to stabilize the vasculature architecture, whereupon blood begins to flow through the new blood vessels.
Angiogenesis, the process of growth of new blood vessels, is an essential process in wound healing and for restoring blood flow to tissues after injury. The discovery of growth factors that stimulate this process has had a major influence on medical treatment of crippling and life-threatening conditions resulting from loss of blood circulation. At least 20 growth factors have been identified that stimulate angiogenesis. The growth factor most widely studied, and used clinically, is the pro-angiogenic platelet-derived growth factor-BB (PDGF-BB). PDGF is released from many cell types including activated platelets, activated macrophages, endothelial cells, fibroblasts and tumor cells, and PDGF was approved by the FDA in December 1997 for clinical use as a topical agent for diabetic foot ulcers. A second growth factor developed for clinical use is the vascular endothelial growth factor (VEGF). Growth factors of this type and biologically active analogs are typically mid-sized proteins which can be produced by recombinant techniques (e.g. in yeast), and activation of angiogenesis by growth factors is accomplished, at least in part, by stimulation of cytokine production.
IL-8 is a cytokine that activates neutrophils and has potent chemotactic activity on neutrophils and lymphocytes. The inflammatory event at the site of infection or injury activates monocytes and macrophages, which release IL-8. Inflamed endothelial tissue also releases IL-8, which attracts neutrophils from blood into the tissue during the initial phase of the defense mechanism. The consequence is a vicious cycle of recruitment of neutrophils in response to IL-8, damage to tissues, and more production of IL-8 leading to deleterious inflammation as a side effect. In addition, neutrophils adhere to inflamed endothelial tissues through integrins secreted from the cells, and ICAM-1 can stimulate release of the integrins to which neutrophils bind, thereby increasing the level of deleterious inflammation at the site of injury even further. Increased levels of certain types of clinically deleterious cytokines, such as IL-8 and ICAM-1, at a site of infection or injury can therefore cause deleterious side effects which can hinder the process of healing.
The healing of wounds in mammalian tissue may be enhanced by the application, either alone or in combination with a cytokine and/or growth factor, of certain neuropeptides such as Tachykinins, Substance P, Substance K, and the like as well as calcitonin gene-related peptides. The use of such peptides for clinical applications has, however, been hampered by several problematic issues including deleterious side effects. Substance P, for example, is a known mediator of pain impulses and its effects on wound healing have been known for several years. However, Substance P has also been shown to stimulate neurons to release factors that recruit inflammatory cytokines and neutrophils to the site of a wound, thereby causing pain and inflammation.
Therefore, the use of peptides or growth factors and their analogs as therapeutic agents for wound healing can be problematic for a number of reasons, including efficacy, cost, and deleterious side effects such as inflammation. Information relevant to attempts to address one or more of these problems can be found in the following references: U.S. Pat. No. 7,105,481, U.S. Patent Application No. 2007/0021342, and U.S. Patent Application No. 2007/0154448. However, each one of these references suffers from one or more of the following disadvantages:
1. the requirement for expression of a polynucleotide containing the nucleotide sequence encoding the protein, which can complicate production and significantly increase costs;
2. the requirement for purification of expressed proteins from the other proteins of the host cell, which can complicate production and significantly increase costs;
3. administration of angiogenic growth factors without the further ability to activate phagocytes and thereby enhance efficacy or ameliorate infection or other concomitant disorders;
4. administration of angiogenic growth factors without the further ability to reduce inflammation, thereby reducing deleterious side effects; and
5. administration of a peptide which can stimulate the release of factors that recruit inflammatory cytokines and neutrophils to the site of a wound, causing pain and inflammation.
Therefore, in light of the available treatments for promoting wound healing by stimulating angiogenesis, there is a need to provide practical, cost-effective therapies that enhance or optimize chronic wound healing without causing deleterious side effects.